Project Summary Adjuvants are essential components of vaccines, and function to enhance the immune response to a co-administered antigen. Adjuvants are a primary focus of modern vaccine development due to the fact that despite decades of research only two adjuvants, alum and monophosphoryl lipid A (MPLA), are clinically approved for human use. While these adjuvants are effective in stimulating immune responses for some vaccines, their ability to potentiate an immune response for drugs of abuse vaccines has achieved mixed results. As a general approach, clinical translation of vaccines for drugs of abuse has been stunted by variability in broad patient populations, and we hypothesize that poorly immunogenic adjuvants are the reason. Currently, there are no adjuvants that effectively elicit potent immune responses to drugs of abuse and until new adjuvants are discovered, successful translation of active vaccination to drugs of abuse will not be possible. In the our on-going effort to deliver clinically-viable vaccines for heroin, oxycodone, methamphetamine (METH), and nicotine, the Janda Laboratory has investigated 4-((2-formyl-3-hydroxyphenoxy)methyl)benzoic acid (tucaresol) as a synthetically-tractable adjuvant. Recently, we reported the first liposomal formulation of tucaresol, termed [L(LT1)]. We used this adjuvant in a vaccine for METH and demonstrated high antibody titers in a mouse model. Despite our initial success, our understanding of how [L(LT1)] stimulates the immune system remains limited. In this proposal, I will elucidate the mechanism of [L(LT1)] immunopotentiation. My approach will include investigating the induction of Th-cell proliferation, potential covalent chemical ligation of Th cell surface amines, and cellular localization of [L(LT1)] (Aim 1) and evaluating a series of [L(LT1)] analogues in rodents for their ability to stimulate an immune response (Aim 2). In Aim 2, this previously- constructed SAR series will dissect how tucaresol?s chemical makeup plays a role in immunostimulation. The proposed work will help to achieve our long-term goal of delivering clinically-relevant vaccines for drugs of abuse. The work proposed here is innovative and impactful because it marks the first detailed, mechanistic study of immunopotentiation by [L(LT1)]. These pioneering studies will have a lasting impact because they provide a foundation for the future development of [L(LT1)] analogues as next-generation vaccine adjuvants. Importantly, we envision that liposomal tucaresol offers a new adjuvant platform with broad applicability to vaccines for all drugs of abuse. This work is directly aligned with the mission of NIDA to support research aimed at improving the treatment of substance use disorders.